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Methods for designing specific ion channel blockers

a technology of ion channel blocker and design method, which is applied in the direction of immunoglobulins, peptides, antibody medical ingredients, etc., can solve the problems of lack of specificity, lack of functional redundancy, complicated results interpretation, etc., and achieves rapid and effective means

Inactive Publication Date: 2005-12-08
HUANG XIN YUN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] A number of compounds useful in treating various diseases in animals, including humans, are thought to exert their beneficial effects by modulating the functioning of ion channels. An understanding of the pharmacology of compounds that interact with ion channels, and the ability to rationally design compounds that will interact with ion channels to have desired therapeutic effects, have been hampered by the lack of rapid, effective means to identify those compounds which interact with specific ion channels. The availability of rapid, effective means to identify compounds which interact with ion channels would enable the rapid screening of a large number of compounds to identify those candidates suitable for further, in-depth studies of therapeutic applications. With the method of the present invention, blockers for each specific ion channel based on available nucleotide or amino acid sequence information are rationally designed. Using a specific antibody against the external vestibule of a channel protein, the specific blocking of this channel is achieved by this antibody, but other related channels are not blocked. This approach provides a method to rationally design specific ion channel blockers.

Problems solved by technology

Although genetic manipulation with targeted deletion of ion channel genes would be helpful, the interpretation of results could be complicated by functional redundancy and developmental abnormalities.
Some ion channel blockers are available, but they usually affect a group of ion channels and, thus, lack specificity towards one specific channel protein.

Method used

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  • Methods for designing specific ion channel blockers
  • Methods for designing specific ion channel blockers
  • Methods for designing specific ion channel blockers

Examples

Experimental program
Comparison scheme
Effect test

example 1

Suppression of Kv1.2 Current by Kv1.2-BA Antibody

[0055] To test whether antibodies specific for peptides around the pore-regions of individual ion channels can be used as a channel blocker, the blocking ability of an affinity-purified polyclonal antibody generated against a 15 amino-acid peptide of a delayed-rectifier potassium channel Kv1.2 (Huang, X. Y. et al., Cell 75:1145-1156 (1993)) (FIG. 1A) was examined. This sequence identified as SEQ ID NO:1:

Phe Ala Glu Ala Asp Glu Arg Asp Ser Gln Phe Pro Ser Ile Pro1               5                   10                  15

located between the S5 transmembrane and the pore-forming (P) region, is very likely part of the external vestibule (or the outer mouth) of the channel protein (FIG. 1B) (Lu, Q. et al., Science 268:304-307 (1995); Hidalgo, P. et al., Science 268:307-310 (1995); Aiyar, J. et al., Neuron 15:1169-1181 (1995), which are hereby incorporated by reference). Kv1.2 was stably expressed in a mammalian cell line, HEK-293 cells ...

example 2

[0056] To ensure that the Kv1.2-BA antibody does indeed bind to the external region of Kv1.2 channel protein and that the blocking effect is due to the binding of the Kv1.2-BA antibody to the channel protein, Kv1.2-BA antibody was preincubated with the immunogenic peptide that was used to generate Kv1.2-BA. If blocking is due to binding of the antibody to the peptide sequence in the external vestibule of the channel protein, preincubation with the peptide should prevent the inhibition. As shown in FIG. 2E, addition of 250 nM Kv1.2-BA after preincubation with the immunogenic peptide only produced about 25% inhibition. Preincubation with a control peptide did not inhibit the Kv1.2-BA-induced suppression of Kv 1.2 currents (FIG. 2E). Thus, the majority of the inhibition by Kv1.2-BA on Kv1.2 currents is due to specific finding of Kv1.2 to the particular peptide sequence around the pore region of Kv1.2 channels. The residual about 25% inhibition is likely due to either nonspecific blocki...

example 3

[0057] No Effect of Kv1.2 Current by a Control Antibody Kv1-NA

[0058] To further exclude the possibility that the Kv1.2 BA blocking effects were non-specific and that any antibody added outside cells somehow interferes the channel function, the effects on the Kv1.2 currents by another affinity-purified polyclonal antibody (Kv1-NA) was examined (FIG. 3A and 3B). Kv1-NA was generated against a peptide having SEQ ID NO:2:

Asp Pro Leu Arg Asn Glu Tyr Phe Phe Asp Arg Asn Arg Pro Ser1               5                   10                  15

from the intracellular N-terminus of Kv1.2, which is identical in all members of the Kv1 family (FIG. 1D). Addition of this control antibody Kv1-NA had no effect at low concentrations (100 nM) on the Kv1.2 currents (FIG. 3B). These results indicate that the effect of antibody Kv1.2-BA is specific.

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Abstract

The present invention relates to a method of designing an ion channel blocker for an ion channel which includes providing an ion channel having an external vestibule portion and raising an antibody, binding portion, probe, or ligand specific to the external vestibule portion of the ion channel, where the antibody, binding portion, probe, or ligand inhibits ion transport through the ion channel. The present invention further relates to a method of inhibiting ion transport through an ion channel, a method for screening a drug for effectiveness as an ion channel blocker, and an antibody, binding portion, probe, or ligand.

Description

[0001] This application is a divisional of U.S. patent application Ser. No. 09 / 273,217, filed Mar. 19, 1999, which is hereby incorporated by reference in its entirety and claims the benefit of U.S. Provisional patent application Ser. No. 60 / 079,268, filed Mar. 25, 1998, which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION [0002] Cell membranes must allow passage of various polar molecules, including ions, sugars, amino acids, and nucleotides. Special membrane proteins are responsible for transferring such molecules across cell membranes. These proteins, referred to as membrane transport proteins, occur in many forms and in all types of biological membranes. Each protein is specific in that it transports a particular class of molecules (such as ions, sugars, or amino acids) and often only certain molecular species of the class. All membrane transport proteins that have been studied in detail have been found to be multipass transmembrane proteins. By f...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K14/705
CPCA61K2039/505C07K14/705C07K16/28
Inventor HUANG, XIN-YUN
Owner HUANG XIN YUN
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